Metazoan cell types vary widely in size, and the basis for this is not understood. Proliferating populations of cells show constant variation around the mean size, and cell size changes in several pathologies including cancer. It was unclear until recently whether there are processes that directly control cell size. We developed methods for measuring dry mass and dry mass changes over time in single cells, revealing a discrete feedback between size and growth rate in proliferating cells around the G1/S transition. We now propose to find the molecular basis for size control in proliferating cells and to extend this inqury to the effect of growth and density on size control, and how size control drives morphogenesis in intact tissues. We first ask whether the G1/S feedback we identified operates on protein synthesis or degradation, using Stimulated Raman Spectroscopy (SRS) adapted to the microscope and Quantitative Phase Microscopy. We will perform single cell transcriptional profiles on cells of varying size and cell cycle stage to look for genes that correlate with the activation of size control, and perturb size, growth, and cell cycle with drugs and inhibitors, measuring effects on size and on variance in size. Candidate genes will be tested by knockdown and overexpression. Based on our recent findings that the hippo pathway affects cell density-dependent size control, we will examine other cell circuits affected, using mass spectrometry, reverse phase arrays and single cell transcriptomics. In a striking process of natural hypertrophy, we have shown that chondrocytes undergo discrete phases of balanced growth, swelling, and further balanced growth at lower mass density. To probe the circuits underlying this process, we will isolate chondrocytes from different hypertrophic regions of a single growth plate and in different growth plates with different elongation kinetics, and profile single cell transcriptomes. Our overall goal is to identify the circuits that underlie size feedbac in proliferating cells, to determine if similar feedbacks exist in non-dividing cells, and to understand how size control integrates with growth and proliferation in developing systems. This will provide a basis for understanding size control in cell differentiation and homeostasis, and the role of hypertrophy in human development and pathology.